Transplantation for organ failure is limited by the problems of organ scarcity and the need for lifelong immunosuppression. Tissue engineering holds the promise to create new organs outside the human body but has been hindered by 1.) the inability to adequately vascularize tissue constructs and 2.) the inability to efficiently re-integrate these tissues into the systemic circulation. Classical approaches to tissue engineering using cells seeded onto resorbable matrices have had success in replicating simple structures but have been unable to create complex parenchymal organs because of the difficulty in creating patterned vascular networks. To solve this problem, we have developed a novel approach to engineer constructs of organ-level complexity by using pre-existing, explanted microcirculatory beds as the scaffold for tissue engineering. Since this approach starts with the vascular network as a foundation, it builds autologous tissue from the inside-out, in a manner similar to embryonic development or stem cell mediated tissue regeneration. During our previous NIH funding period, we demonstrated an ability to sustain explanted vascular beds for extended periods ex vivo (48-72hrs), genetically modify their growth milieu, and efficiently seed them with progenitor cells creating functional neo-organ units for re-implantation. Based on this success, we postulate that more prolonged cultivation of explanted microcirculatory beds will permit generation of autologous vascularized neo-organs which can reliably substitute a functional or physiologic role for a failing organ. In this proposal, Specific Aim 1 will define the perfusion conditions necessary for reliable viability, growth, and directed manipulation of explanted microcirculatory beds for 3-14 days ex vivo. In Specific Aim 2, the optimal seeding and differentiation conditions of infused progenitor cells to generate vascularized neo-organs will be determined. Finally, Specific Aim 3 will investigate the durability and functional capacity of re-implanted vascularized neo-organ units to fulfill a physiologic role in vivo. We believe this inside-out approach to tissue engineering will facilitate the generation of vascularized organ-level constructs by specifically addressing the critical issues that preclude success in other tissue engineering paradigms. PUBLIC HEALTH RELEVANCE: Tissue engineering holds the promise to create new organs but progress has been limited by poor vascularization of tissue constructs and ineffective re-integration of these tissues into the systemic circulation. To address this problem, we have developed a novel approach to engineer organ-level constructs by using pre-existing, explanted microcirculatory beds as an autologous scaffold for tissue regeneration. Our initial results demonstrate that viable microcirculatory beds can be maintained ex vivo, seeded with progenitor cells, and transfected to produce therapeutic peptides - all critical initial steps toward generating vascularized organ- level constructs.